Inhibitor of differentiation protein-2 (Id2) is a dominant negative helix-loop-helix (HLH) protein, and a positive regulator of proliferation, in various cells. The N-terminal region of Id2 contains a consensus cdk2 phosphorylation sequence SPVR, which may be involved with the induction of apoptosis, at least in myeloid 32d.3 cells. However, the role of Id2 phosphorylation at serine 5 in skeletal muscle cells is unknown. The objective of this study was to determine if the phosphorylation of Id2 at serine 5 alters its cellular localization and its role in apoptosis in C2C12 myoblasts. Overexpression of wild type Id2 decreased MyoD protein expression, which corresponded to the increased binding of Id2 to basic HLH proteins E47 and E12. Bromodeoxyuridine incorporation was significantly decreased by the overexpression of phospho-ablated Id2 (S5A); conversely, overexpression of wild type Id2 increased cellular proliferation. The subcellular localization of Id2 and phospho-mimicking Id2 (S5D) were predominantly nuclear compared to S5A. The decreased nuclear localization of S5A corresponded to a decrease in cellular proliferation, and an increase in apoptosis. These data suggest that unphosphorylated Id2 is primarily localized in the cytosol, where it is growth suppressive and potentially pro-apoptotic. These results imply that reducing unphosphorylated Id2 may improve the pool of myoblasts available for differentiation by increasing proliferation and inhibiting apoptosis.